Chimeraplasty concerns the introduction of directed alterations in a specific site of the DNA of a target cell by introducing duplex oligonucleotides, which are processed by the cell""s homologous recombination and error repair systems so that the sequence of the target DNA is converted to that of the oligonucleotide where they are different. The present invention concerns a chimeraplasty method that is practiced in a cell-free system.
Chimeraplasty
Chimeraplasty in eukaryotic cells and duplex recombinagenic oligonucleotides for use therein are disclosed in U.S. Pat. No. 5,565,350, issued Oct. 15, 1996, and No. 5,731,181, issued Mar. 24, 1998 by E. B. Kmiec (collectively xe2x80x9cKmiecxe2x80x9d). The recombinagenic oligonucleotides disclosed by Kmiec contained ribo-type, e.g., 2xe2x80x2-O-methyl-ribonucleotides, and deoxyribo-type nucleotides that were hybridized to each other and were termed Chimeric Mutational Vectors (CMV). A CMV designed to repair a mutation in the gene encoding liver/bone/kidney type alkaline phosphatase was reported in Yoon, K., et al., 1996, Proc. Natl. Acad. Sci. 93, 2071. The alkaline phosphatase gene was transiently introduced into CHO cells by a plasmid. Six hours later the CMV was introduced. The plasmid was recovered at 24 hours after introduction of the CMV and analyzed. The results showed that approximately 30% to 38% of the alkaline phosphatase genes were repaired by the CMV.
A CMV designed to correct the mutation in the human xcex2-globin gene that causes Sickle Cell Disease and its successful use was described in Cole-Strauss, A., et al., 1996, Science 273, 1386. A CMV designed to create a mutation in a rat blood coagulation factor IX gene in the hepatocyte of a rat is disclosed in Kren et al., 1998, Nature Medicine 4, 285-290. An example of a CMV having one base of a first strand that is paired with a non-complementary base of a second strand is shown in Kren et al., June 1997, Hepatology 25, 1462.
U.S. patent application Ser. No. 08/640,517, filed May 1, 1996, by E. B. Kmiec, A. Cole-Strauss and K. Yoon, published as WO97/41141, Nov. 6, 1997, and application Ser. No. 08/906,265, filed Aug. 5, 1997, disclose methods and CMV that are useful in the treatment of genetic diseases of hematopoietic cells, e.g., Sickle Cell Disease, Thalassemia and Gaucher Disease.
An example of the use of a CMV having one base of a first strand that is paired with a non-complementary base of a second strand is shown in Kren et al., June 1997, Hepatology 25, 1462. In Kren, the strand having the different desired, sequence was the strand having 2xe2x80x2-O-methyl ribonucleotides, which was paired with the strand having the 3xe2x80x2 end and 5xe2x80x2 end. U.S. Pat. No. 5,565,350 described a CMV having a single segment of 2xe2x80x2-O-methylated RNA, which was located on the chain having the 5xe2x80x2 end nucleotide.
Applicants are aware of the following provisional applications that contain teaching with regard to chimeric mutational vectors: By Steer et al., Serial No. 60/045,288 filed Apr. 30, 1997; Serial No. 60/054,837 filed Aug. 5, 1997; Serial No. No. 60/064,996, filed Nov. 10, 1997; and by Steer and Roy-Chowdhury et al., Serial No. 60/074,497, filed Feb. 12, 1998, entitled xe2x80x9cMethods of Prophylaxis and Treatment by Alteration of APO B and APO E Genes.xe2x80x9d
Cell-Free Recombination
Various reports of homologous recombination using a cell-free extract have been published.
Hotta, Y., et al., 1985, Chromosoma 93, 140-151 report the use of an extract of yeast, mouse spermatocytes and Lilium to effect homologous recombination between two mutant pBR322 plasmids. One of the plasmids was supercoiled, the second plasmid could be linearized or supercoiled. The maximum rate of recombination was less than 1%. A similar experiment using mutant defective pSV2neo and extracts of EJ cells was reported in Kucherlapati, R. S. et al., 1985, Molecular and Cellular Biology 5, 714-720. The maximum rate of recombination was about 0.2%. Kucherlapati reported an absolute requirement that one of the mutant plasmids be linearized. In contrast Hotta, reported recombination between two circular plasmids, although the rate of recombination between circular and linear plasmids was higher.
The report of Jessberger, R., and Berg, P., 1991, Mol. and Cell. Biol. 11, 445 concerns recombination catalyzed by nuclear extracts between plasmids. It stands in contrast to both of the above in two respects. The rate of recombination reported was about 20%, in contrast to rates of less than 0.5%. In addition Jessberger observed the same rate of recombination between circularized plasmids as between a circularized and a linear plasmid.
A related experiment using human nuclear extracts was reported by Lopez, B. S., et al., 1992, Nucleic Acids Research 20, 501-506. Lopez reported recombination in a cell-free system between a linearized plasmid and an unrelated supercoiled plasmid that is not viable in the subsequent selection conditions. The linearized and supercoiled plasmid each contain a lacZ gene; which is a mutant in the linearized plasmid. The linearized plasmid is cut in the lacZ gene at a variable distance from the mutation. Homologous recombination between the site of the mutation and the cut, accordingly, results in the circularization of the plasmid that then becomes viable and the gain of lacZ function. Lopez reports no detectable homologous recombination when the cut and the mutation were 15 base pairs apart. Homologous recombination at a low level was observed when that distance was 27 base pairs. No further increase in the rate of homologous recombination was observed when the distance was made greater than 165 base pairs. Lopez et al., 1987, Nucleic Acids Research
Rad51 and Rad52 Activity in Recombination
Homologous recombination is the process whereby the genes of two chromosomes are exchanged. The rate of homologous recombination between two genetic loci is inversely proportional to their genetic linkage, tightly linked genes rarely recombine. In addition to its genetic function homologous recombination allows a somatic cell to repair DNA damaged by double strand breaks.
The first step in homologous recombination is believed to be synapse formation. A synapse is a DNA molecule in which one chain is hybridized to two other chains. Synapse formation requires an enzymatic activity and energy input from ATP hydrolysis. An artifactual assay in a cell-free system for the enzymatic activity believed to be required for synapse formation is xe2x80x9cstrand transfer.xe2x80x9d In a typical strand transfer assay a circular single strand DNA is combined with a linear duplex to produce a xe2x80x9cnickedxe2x80x9d or relaxed circular duplex and a linear single strand. The Rad51 gene from yeast, mice and humans has been cloned and catalyzes strand transfer. Rad51 is believed to participate in synapse formation. Baumann, P., et al., 1996, Cell 87, 757-766; Gupta, R. C., 1997, Proc. Natl. Acad. Sci. 94, 463-468. The strand transfer activity is further enhanced by the presence of Rad52 protein and replication protein A. Baumann, P., and West, S.C., 1997, EMBO J. 16, 5198-5206; New, J. H., et al., 1998, Nature 391, 407-410; Benson, F. E., et al., 198, Nature 391, 401-404. Although RAD51 protein unlike Rec A binds to duplex DNA, Baumann and West op cit.; Benson, F. E., et al., EMBO J., 13, 5764-5771, in the presence of RAD52, its binding is directed toward single stranded DNA.
In yeast, Rad51 or Rad52 defective individuals are radiation sensitive because of an inability to repair double strand breaks. In mice, Rad51 knock out results in embryonic leathality. Tsuzuki, T., et al., Proc. Natl. Acad. Sci. 93, 6236-6240; Lin, S. D., and Hasty, P. A., Mol. Cell. Biol., 16, 7133.
Cell-Free Mismatch Repair
The intrinsic (thermodynamic) fidelity of DNA replication would lead to an unacceptably high rate of mutation without the presence of an xe2x80x9cerror correctingxe2x80x9d mechanism. Mismatch repair is one such mechanism. In mismatch repair, duplex DNA having a base paired to a non-complementary base is processed so that one of the strands is corrected. The process involves the excision of one of the strands and its resynthesis. Reports of mismatch repair in cell-free eukaryotic systems can be found in Muster-Nassal and Kolodner, 1986, Proc. Natl. Acad. Sci. 83, 7618-7622 (yeast); Glazer, P. M., et al., 1987, Mol. Cell. Biol. 7, 218-224 (HeLa cell); Thomas D. C., et al., 1991, J. Biol. Chem., 266, 3744-3751 (HeLa cell); Holmes et al., 1991, Proc. Natl. Acad. Sci., 87, 5837-5841(HeLa cell and Drosophila). The HeLa and Drosophila cell-free systems required that one strand of the mismatched duplex be nicked for full activity. By contrast, reports of repair in Xenopus egg extracts did not require that the mismatched duplex be nicked. Varlet, I., et al., 1990, Proc. Natl. Acad. Sci. 87, 7883-7887. However, in Varlet the mismatch was repaired in a random fashion, i.e., the strands acted as templates with equal frequency.
Many of the genes required for mismatch repair in yeast and humans have been cloned based on homology with the E. coli mismatch repair genes. Kolodner, R., 1996, Genes and Development 10, 1433-1442. Cells having defective mismatch repair genes show genetic instability, termed Replication Error (RER), particularly evident in microsatellite DNA, and malignant transformation. Extracts of RER cells do not have mismatch repair activity. Umar, A., et al., J. Biol. Chem. 269, 14367-14370.